Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system

Hiroaki Mon, Jaeman Lee, Mai Fukushima, Yudai Nagata, Mie Fujii, Jian Xu, Oumi Nishi, Kazuhiro Iiyama, Takahiro Kusakabe

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

Abstract

Mutation and polymorphism detection by nucleases has become a more important tool in clinical and biological researches. There are several kinds of single-stranded nucleases for detecting mismatched DNAs. One of them, CEL II, was isolated from Apium graveolens and cleaves DNA with high specificity at sites of mismatch. High-throughput mutation scanning requires large quantity of CEL II endonuclease. Here, we demonstrate high-level expression of CEL II using silkworm-baculovirus system. The recombinant CEL II secreted in silkworm hemolymph was glycosylated and susceptible to N-glycosidase F. Additionally, larger metal ions such as Ca2+ and Sr2+ were able to replace Mg2+ and enhanced mismatch cleavage activity of CEL II. These results indicate that the silkworm-baculovirus platform is a good alternative system to obtain the functional CEL II.

Original languageEnglish
Pages (from-to)6813-6822
Number of pages10
JournalApplied Microbiology and Biotechnology
Volume97
Issue number15
DOIs
Publication statusPublished - Aug 1 2013

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology

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